Isoliquiritigenin Alleviates LPS-induced Inflammation Via Modulating Mitochondrial Biogenesis In RAW 264.7 Cells

Background: Isoliquiritigenin(ISL)is a chalcone-like compound found in the homologous plant Glycyrrhiza uralensis Fisch.It has been found to have excellent antioxidant,anti-inflammatory and anti-tumor activities,however,the exact biological mechanism of its anti-inflammatory activity is still unclear.Objective: To investigate the effect of ISL on the inflammatory response in LPSinduced RAW 264.7 macrophages,clarify its mechanism of action from the perspective of mitochondrial function and biosynthesis,and to provide a new basis for the traditional application of licorice.Methods:(1)MTT assay was used for detection of cell viability;(2)Optical microscopy was used for observation of morphology;(3)Griess assay was used for detection NO content and ELISA assay was used for detection the level of TNF-α in cells;(4)The expression levels of inflammation-related protein i NOS,oxidative stressrelated protein Nrf2,apoptosis-related protein(Bcl2,Bax)and mitochondrial autophagy(Parkin,Pink1)were measured by Western blotting;(5)DCFH-DA,MitoSOX,JC-1 and Mito-Tracker Green staining were used for observation of the total reactive oxygen species,mitochondrial-derived ROS,mitochondrial membrane potential level and mitochondrial mass via flow cytometry;(6)Fluorescence enhancement method based on luciferase was used to detect ATP content;(7)Mitochondrial DNA damage was determined by Long PCR;(8)The expression levels of mitochondrial dynamics related proteins(Drp1,OPA1,Mfn1/2)and mitochondrial biogenesis(PGC-1α,NRF1,Tfam)were measured by Western blotting.Results:(1)ISL inhibited the proliferation of RAW 264.7 macrophages in a dosedependent manner,while ISL below 20 μmol/L showed no obvious cytotoxic activity;(2)LPS induced RAW 264.7 macrophages to differentiate into inflammatory cells,and the cells differentiate from a round shape in a normal state to a typical spindle shape;(3)Compared with the blank control group,the expression levels of pro-inflammatory factors NO,TNF-α and i NOS protein in the model group were significantly increased,while ISL treatment significantly reduced NO,TNF-α and i NOS levels.(4)Isoliquiritin significantly increased the level of Nrf2 protein and the ratio of Bcl-2/Bax in RAW264.7 cells,and reversed the LPS-induced down-regulation of Parkin protein expression in a concentration-dependent manner.(5)ISL improved the mitochondrial disorder in LPS-induced RAW 264.7 macrophages,increased the rate of mitochondrial ATP synthesis,and maintain the normal membrane potential of mitochondria.(6)LongPCR and Western blotting results showed that LPS caused mitochondrial DNA damage and abnormal expression of mitochondrial biogenesis-related proteins;and ISL inhibited mitochondrial damage and restored the expression level of mitochondrial synthetic protein,improved mitochondrial function,and maintained mitochondrial homeostasis.Conclusion: LPS induced RAW 264.7 macrophages transform to M1 proinflammatory and release pro-inflammatory factors such as NO,TNF-α and i NOS;ISL treatment significantly inhibited LPS-induced RAW 264.7 macrophages transform to M1 conversion of,and down-regulating NO and TNF-α level.In LPS-induced RAW264.7 macrophages,different concentrations of ISL significantly inhibited the production of total cell ROS and mitochondrial-derived ROS,improved cell oxidative stress caused by LPS,and prevent cell damage;in addition,ISL also improved mitochondrial function,inhibited LPS-stimulated RAW 264.7 cells apoptosis and mitochondrial autophagy.